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Applikationshinweise
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Example of Immunohistochemistry application Perfusion protocol for Adult male monkeys (Macaca fascicularis)(weight 3-3.5 kg). 1-The animals can be deeply anaesthetized with ketamine (8mg/kg, intramuscular) and sodium thiopental (500 mg/kg, intraperitoneal). 2-Heparinized, and perfused via the ascending aorta with 300 ml of cold physiologic saline (0.9% NaCl) and with the following fixative solutions:a) 500 ml of 1% paraformaldehyde in 0.1 M phosphate-buffer (PB), pH 7.2, at room temperature (two minutes). b) 2,500 ml of 4% paraformaldehyde in 0.1 M PB, pH 7.2, at 4ºC (ten minutes). c) 5,000 ml of cold 4% paraformaldehyde in 0.1 M PB, pH 7.2 (fifty minutes). d) 2,000 ml of cold 5% sucrose in 0.1M PB, pH 7.2 (twenty minutes). d) Dissect out the brains and place in 10% glycerol and 2% dimethylsufoxide (DMSO) in 0.1M PB, pH 7.2, at 4ºC for two days, and finally keep at the same temperature in 20% of glycerol and 2% DMSO in PB until the brains will be cut on a freezing microtome. Around 50 µm-thick serial sections will be obtained, kept at 4º C in PB (0.1 M, pH 7.2) containing 20% of glycerol and 30% of ethylene glycol, and processed for immunostaining. Example of immunohistochemical protocol 1-In order to avoid possible interference with endogenous peroxidase, free-floating sections will be treated with distilled water containing NH 3 (20%), H 2 O 2 (30%) and NaOH (1%) for 20 min (other method is using a solution with 33% of H 2 O 2 and 66% of methanol). 2-Then, wash the sections for 20 min in 0.15 M phosphate-buffered saline (PBS) (pH 7.2) 3-Pre-incubate for 30 min in PBS containing 10% of normal horse serum and 0.3% of Triton X-100 (mixed solution). 4-Incubate at room temperature (1h 30min) and overnight at 4º C in the same mixed solution containing Riboflavin antiserum (diluted 1/5001/1,000 as recommended dilutions). 5-Then, the sections will be wash in PBS (30 min). 6-After that we will incubate for 60 min at room temperature with biotinylated anti-rat immunogammaglobulin (Vector) diluted 1/200 in PBS. 7-Wash during 30 min with PBS. 8-Sections will be incubated for 1 h with a 1/100 diluted avidin-biotin-peroxidase complex (Vectastain). 9-After that we will wash the sections in PBS (30 min) 10-Wash with Tris-HCl buffer (pH 7.6)(10 min). 11-The tissue-bound peroxidase will be developed with H 2 O 2 using 3, 3' diaminobenzidine as chromogen. 12-Finally the sections will be rinsed with PBS and coverslipped with PBS/Glycerol (1/1). Recommended dilutions for Immunohistochemistry(1/500-1/1,000) Recommended dilutions for Western Blot (1/500-1/1,000)
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